Reference control containing a nucleated red blood cell component

ABSTRACT

A method of making a reference control containing a nucleated red blood cell component includes providing a blood cell containing a nucleus; treating the blood cell with a treatment solution to alter a nucleus property from a natural value to a target value suitable for simulating nucleated red blood cells on a blood analyzer; and suspending treated blood cell in a suspension medium to form a reference control. The method also includes integrating the nucleated red blood cell component with white blood cell, red blood cell, platelet and reticulocyte components. Further disclosed is a cell treatment composition for altering a nucleus property, which includes a conditioning component, a lytic component for permeating cell membrane, and a fixing component for preserving the cell nucleus. Also disclosed is a method of using the reference control for measurement of nucleated red blood cells on a blood analyzer.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of patent application Ser. No.11/094,644, filed on Mar. 30, 2005, now U.S. Pat. No. 7,135,341 issuedon Nov. 14, 2006, which claims the benefit under 35 USC 119 (e) of theprovisional patent application Ser. No. 60/560,236, filed on Apr. 7,2004. Both parent applications are herein incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to a reference control compositioncontaining a nucleated red blood cell component and the method of makingand using the reference control composition for determination ofnucleated red blood cells of a blood sample on a blood analyzer.

BACKGROUND OF THE INVENTION

Quality control has long been a necessary and routine procedure inclinical hematology. Accuracy in the counting of various types of bloodcells is dependent, in part, upon the use of adequate control productsand methods of using the control products. With the numerous types ofequipment for particle counting now available, quality control by theuse of control products is necessary, since the possibility of aninstrument malfunctioning is ever present. The traditional method ofmaintaining a quality control program for automatic particle countingequipment has consisted of providing fresh human blood as a whole bloodstandard. However, this fresh blood is usable for only one day,therefore, various manufactured control products which have longerproduct lifetime have been developed.

Commonly used particles in a control product simulate or approximate thetypes of particles or cells that are intended to undergo analysis.Consequently, these particles have been frequently referred to as analogparticles. The analog particles should be selected or designed so thatthey have certain characteristics that are similar to those of theparticles or cells to be analyzed in the instruments. Exemplarycharacteristics and parameters include similarities in size, volume,surface characteristics, granularity properties, light scatteringproperties and fluorescence properties.

Various commercial reference control products are now available, whichuse various processed or fixed human or animal blood cells as analogs ofhuman blood cells. U.S. Pat. No. 5,512,485 (to Young et al) teaches ahematology control comprising several white blood cell analogs made ofprocessed and fixed animal red blood cells. These fixed red blood cellsare fixed at their near native cellular size for simulating specificwhite blood cell subpopulation in a blood sample. Commercially availablehematology controls can also contain red blood cell, platelet,reticulocyte and nucleated red blood cell components.

Nucleated red blood cells (NRBCs), or erythroblasts, are immature redblood cells. They normally occur in the bone marrow but not inperipheral blood. However, in certain diseases such as anemia andleukemia, nucleated red blood cells also occur in peripheral blood.Therefore, it is of clinical importance to measure NRBCs in peripheralblood. In recent years, several detection methods for measuringnucleated red blood cells in a blood sample on a hematology instrumenthave been reported. U.S. Pat. No. 5,559,037 (to Kim et al.) discloses amethod for flow cytometric analysis of nucleated red blood cells andleukocytes. The method uses fluorescence, low angle light scatter andaxial light loss measurements to differentiate NRBCs from white bloodsamples. U.S. Pat. No. 5,879,900 (to Kim et al) further discloses amethod of differentiating NRBCs, damaged white blood cells (WBC), WBCand a white blood cell differential in a blood sample by flow cytometry.

U.S. Pat. Nos. 5,874,310 and 5,917,584 (to Li et al) disclose a methodof differentiating nucleated red blood cells by measuring two angles oflight scatter signals of a blood sample. U.S. Pat. Nos. 5,874,310 and5,917,584 further disclose a method of differentiating nucleated redblood cells by measuring light scatter and DC impedance signals. U.S.Pat. Nos. 6,410,330 and 6,673,618 (to Li et al) disclose a method ofdetermining NRBC by using DC impedance measurement. U.S. Pat. No.6,472,215 (to Huo et al) discloses a method of differentiating nucleatedred blood cells by an impedance measurement in combination with a threedimensional DC, RF and light scatter measurements.

With the development of the above-referenced detection methods fornucleated red blood cells, several hematology controls containing anucleated red blood cell component, or NRBC analog, have been reported.

U.S. Pat. Nos. 6,187,590 and 5,858,790 (to Kim et at) disclose ahematology control comprising a nucleated red blood cell (NRBC) analogmade of lysed and fixed avian or fish red blood cells, or lysed andfixed human lymphocytes. U.S. Pat. Nos. 6,187,590 and 5,858,790 furtherdisclose the method of preparing the NRBC analog, by lysing avian orfish red blood cells with a lysing reagent for 1 to 5 minutes, followedby fixing nuclei from the cells with a fixative at 60 to 70° C. for upto 10 minutes.

U.S. Pat. Nos. 6,406,915, 6,403,377, 6,399,388, 6,221,668 and 6,200,500(to Ryan, et al) disclose a hematology control comprising a NRBC analogderived from avian blood cells. The method includes washing avian redblood cells, such as turkey or chicken red blood cells in a buffersolution and fixing the washed cells with glutaraldehyde phosphatesolution at room temperature for one day. U.S. Pat. No, 6,448,085 (toWang et al) discloses a hematology control comprising a nucleated redblood cell (NRBC) analog which is fixed chicken red blood cells obtainedfrom a commercial source.

U.S. Pat. Nos. 6,653,137 and 6,723,563 (to Ryan) disclose methods ofmaking a nucleated red blood cell component for a hematology control bystabilizing blood cells containing a nucleus, or by lysing and removingcytoplasm from blood cells. U.S. Pat. No. 6,723,563 specifically teachesa method of making the nucleated red blood cell component, whichcomprises the steps of contacting a blood cell which includes a membraneenclosing a nucleus and cytoplasm with a lysing agent for at least 4hours, removing cytoplasm from within the membrane, but preserving thegeneral structure of the membrane about the nucleus. The method furthercomprises fixing the blood cells after removing the cytoplasm.

It has been recognized that the nucleated red blood cell analogsproduced from different cell sources and processed by different methodscan have different properties. For example, the NRBC analog made fromlysed and fixed avian blood cells are suitable for fluorescence basedmeasurement methods, but are too small for sizing methods, such asimpedance or light scatter measurement methods, for the purpose ofsimulating human nucleated red blood cells. Furthermore, the nuclei ofthe stabilized alligator cells without fixation can also be too smallwhen they are analyzed under certain strong lysing conditions used forblood sample analysis on a hematology analyzer. Therefore, it isdesirable to have a method of preparing the NRBC analog which enablesaltering the natural size of cell nucleus of the blood cells used forpreparing the analog, to obtain a target size suitable for apredetermined detection domain for simulating human nucleated red bloodcells.

In terms of cell property manipulation, U.S. Pat. No. 6,146,901 (toCarver et al) discloses a method for manipulating the optical andelectrical properties of a biological particle to achieve selectedtarget values for respective properties. The method includes providing abase biological particle, such as animal red blood cells, having bothoptical and electrical properties at respective natural values;contacting the particles with a preincubation media which includes ahypotonic buffered solution and a polyhydroxy alcohol to manipulate theoptical and electrical properties of the particle; remaining theparticle in the preincubation media for an incubation time; subsequentlycontacting the particle with a primary fixative solution for a period oftime. The incubation time and the time in contact with the primaryfixative solution are selected to manipulate the respective naturalvalues of the optical and electrical properties of the particle toachieve the target values of the optical and electrical properties.Carver et al's method is used to manipulate the cell properties byleaking a quantity of hemoglobin from the red blood cells without lysingthe red blood cells. Carver et al do not teach manipulation oralteration of nucleus properties of a blood cell.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a reference controlcontaining a nucleated red blood cell component. The reference controlcomprises a nucleated red blood cell component obtained by treating ablood cell that contains a nucleus with a treatment solution to alter anucleus property from a natural value to a target value suitable forsimulating nucleated red blood cells of a blood sample on a bloodanalyzer; and a suspension medium suitable for delivering said nucleatedred blood cell component to said blood analyzer for analysis of saidnucleated red blood cells. The nucleus property includes the size, or anoptical property of the nucleus. The optical property can be lightscatter property, axial light loss property, or fluorescence property ofsaid nucleus upon staining with a fluorescence dye.

The reference control can further comprise a white blood cell component,red blood cell component, platelet component, and reticulocytecomponent.

In a further aspect, the present invention is directed to a method ofmaking the reference control containing a nucleated red blood cellcomponent. The method comprises the steps of providing a blood cellcontaining a nucleus; treating said blood cell with a treatment solutionto alter a nucleus property from a natural value to a target valuesuitable for simulating nucleated red blood cells on a blood analyzer,and to preserve said target value; and suspending treated blood cell ina suspension medium to form said reference control.

In another aspect, the present invention is directed to a cell treatmentcomposition for altering a nucleus property. The cell treatmentcomposition comprises a conditioning component, a lytic component forpermeating cell membrane, and enabling said cell treatment compositionbeing in contact with said cell nucleus; a fixing component forpreserving said cell nucleus; wherein said conditioning component, saidlytic component, and said fixing component are present at predeterminedrespective concentrations to alter a nucleus property from a naturalvalue to a target value. The conditioning component comprises a buffer,and an osmolality adjusting agent. The fixing component is a fixativesuch as an aldehyde, oxazolidine, alcohol, cyclic urea, or combinationthereof. The lytic component comprises a quaternary ammonium surfactant.The cell treatment composition can further comprise one or morenon-ionic surfactants.

In yet a further aspect, the present invention is directed to a methodof using a reference control containing a nucleated red blood cellcomponent. The method comprises the steps of providing a referencecontrol containing a nucleated red blood cell component obtained bytreating a blood cell that contains a nucleus with a treatment solutionto alter a nucleus property from a natural value to a target valuesuitable for simulating nucleated red blood cells of a blood sample;providing a blood analyzer adapted for analyzing said blood cell sampleand differentiating nucleated red blood cells from other cell types;passing the control through said blood analyzer for detection of saidnucleated red blood cell component; and reporting nucleated red bloodcells in said reference control. The differentiation of nucleated redblood cells from other cell types is obtained using impedance, oroptical measurement, or combination thereof. The optical measurement canbe fluorescence, light scatter, axial light loss measurements, orcombination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a red blood cell distribution histogram of the washedalligator red blood cells.

FIG. 1B shows a partially displayed white blood cell distributionhistogram of the washed alligator red blood cells.

FIG. 1C shows a DC vs. RLS scattergram of the washed alligator red bloodcells,

FIG. 1D shows a FS (forward light scatter) vs. SS (side scatter)scattergram of the washed alligator red blood cells.

FIG. 1E shows a FL4 vs. FL1 scattergram of the washed alligator redblood cells. It is noted that FL4 and FL1 are in log scale in allfluorescence scattergrams illustrated herein.

FIG. 2A shows a red blood cell distribution histogram of a NRBC analogmade of treated alligator red blood cells using Treatment Solution 1with the process described in Example 1.

FIG. 2B shows a partially displayed white brood cell distributionhistogram of the NRBC analog made of treated alligator red brood cellsusing Treatment Solution 1 with the process described in Example 1.

FIG. 2C shows a DC vs. RLS scattergram of the NRBC analog made oftreated alligator red blood cells using Treatment Solution 1 with theprocess described in Example 1.

FIG. 2D shows the FS vs. SS scattergram of the NRBC analog made oftreated alligator red blood cells using Treatment Solution 1 with theprocess described in Example 1.

FIG, 2E shows the FL4 vs. FL1 scattergram of the NRBC analog made oftreated alligator red blood cells using Treatment Solution 1 with theprocess described in Example 1.

FIG. 3A shows a white blood cell distribution histogram of a clinicalsample containing 27 NRBC/100 WBC.

FIG. 3B shows a DC vs. RLS scattergram of the clinical sample of FIG.3A.

FIG. 3C shows a FL4 vs. FL1 scattergram of a normal whole blood sample.

FIG. 3D shows a FL4 vs. FL1 scattergram of a clinical sample containing13.7 NRBC/100 WBC.

FIG. 4A shows a red blood cell distribution histogram of a NRBC analogmade of treated alligator red blood cells using Treatment Solution 2 asdescribed in Example 2.

FIG. 4B shows a partially displayed white blood cell distributionhistogram of the NRBC analog made of treated alligator red blood cellsusing Treatment Solution 2 as described in Example 2.

FIG. 4C shows a DC vs. RLS scattergram of the NRBC analog made oftreated alligator red blood cells using Treatment Solution 2 with theprocess described in Example 2.

FIG, 4D shows the FS vs. SS scattergram of the NRBC analog made oftreated alligator red blood cells using Treatment Solution 2 asdescribed in Example 2.

FIG. 4E shows the FL4 vs. FL1 scattergram of the NRBC analog made oftreated alligator red blood cells using Treatment Solution 2 asdescribed in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a reference controlcomposition that contains a nucleated red blood cell (NRBC component,and a method of preparing the nucleated red blood cell (NRBC componentand the hematology control composition.

The method includes the steps of: providing a blood cell containing anucleus; treating the blood cell with a treatment solution to alter anucleus property from a natural value to a target value suitable forsimulating nucleated red blood cells of a blood sample on a bloodanalyzer, and to preserve the target value of the nucleus; andsuspending treated blood cell in a suspension medium to form a referencecontrol. For the purpose of the present invention, the nucleated redblood cell component, as well as other cell type components, are alsoreferred to as analogs, for example, NRBC analog. The term “nucleusproperty” used herein includes, but is not limited to, the size, or anoptical property of the nucleus. The optical property can be lightscatter property, axial light loss property, or fluorescence property ofsaid nucleus upon staining with a fluorescence dye.

Suitable examples of blood cells suitable for simulating human nucleatedred blood cells include various nucleated animal red blood cells andsmall mammalian white blood cells. More specifically, reptilian, avianand fish red blood cells, such as alligator, shark and salmon red bloodcells, and mammalian lymphocytes from whole blood or grown in vitro by acell line can be used. In one preferred embodiment, alligator red bloodcells were used for making the nucleated red blood cell component.

In one embodiment, the method of preparing the NRBC analog using animalnucleated red blood cells comprises following process steps:

-   -   1. Collect a quantity of whole blood of a selected animal        species which has nucleated red blood cells in an anticoagulant        containing container. Centrifuge the whole blood and remove the        top layer (including white blood cells, platelets and plasma).    -   2. Wash the packed nucleated red blood cells with a buffered        isotonic wash solution three times.    -   3. Wash the packed nucleated red blood cells with a suspension        medium and re-suspend washed packed cells in the suspension        medium. Preferably, the cell count is in a range from about        0.4×10⁶ to about 0.6×10⁶ cell/μl.    -   4. Treat the re-suspended nucleated red blood cells by adding a        predetermined volume of the re-suspended cells to an equal        volume of a treatment solution; mix well by inversion to form a        cell treatment suspension; and incubate the cell treatment        suspension for a treatment period, to alter a nucleus property        from a natural value to a target value, and to preserve the        target value.    -   6. Separate the treated cells from the cell treatment suspension        by centrifugation.    -   7, Re-suspend the treated cells in a suspension medium suitable        for analysis on a blood analyzer.

One suitable buffered isotonic wash solution is the phosphate bufferedsaline solution (PBS). Various other cell wash solutions known in theart can also be used.

Suitable examples of suspension medium include phosphate buffered salinesolution and an aqueous solution of a plasma substance. As definedherein, an aqueous solution of a plasma substance comprises an aqueoussolution of a serum substance, serum substance in combination with aplasma protein and mixtures thereof. As further defined herein, plasmaprotein comprises one or more of the proteins contained in plasma.Preferably, such plasma proteins comprise albumin, lipoproteins,globulins, fibrinogens and mixtures thereof. These media may containother ingredients known to those skilled in the art to confer cellstability. Example 1 provides three example formulas of the suspensionmedium, Preferably, for long term storage of the NRBC analog, thesuspension medium contains a relatively higher concentration of protein.Other examples of suitable medium are more fully described in U.S. Pat.Nos. 4,213,876, 4,299,726, 4,358,394, 3,873,467, 4,704,364, 5,320,964,5,512,485 and 6,569,682 which are herein incorporated by reference intheir entirety.

The treatment solution, also referred to as a cell treatmentcomposition, for altering a nucleus property for the purpose of thepresent invention comprises a conditioning component, a lytic componentfor permeating cell membrane and enabling the treatment solution to bein contact with the cell nucleus and a fixing component for preservingthe treated nucleus.

In one embodiment, the conditioning component comprises a buffer, and anosmolarity adjusting agent. The buffer and osmolarity adjusting agentcan be two separate chemicals, each providing a different function.However, the buffer and osmolarity adjusting agent can also be the samechemical to provide both functionalities. For example, when a phosphatebuffer is used in the treatment solution, the pair of phosphate saltscan provide both buffering and osmolarity adjusting functions.

In one preferred embodiment, the lytic component comprises a quaternaryammonium surfactant represented by following molecular structure:

wherein R₁ is an alkyl, alkenyl or alkynyl group having 12 to 16 carbonatoms; R₂, R₃ and R₄ are alkyl groups having 1 to 4 carbon atoms and X−is chloride or bromide anion.

Furthermore, the treatment solution can also comprise an ethoxylatedalkyl phenol having an alkyl group with 6 to 12 carbon atoms, andbetween about 10 to about 50 ethylene oxide groups. Moreover, thetreatment solution can further comprise an ethoxylated alkyl alcoholrepresented by following molecular structure:R₁—R₂—(CH₂CH₂O)_(n)—Hwherein R₁ is an alkyl, alkenyl or alkynyl group having 10 to 22 carbonatoms, R₂ is —O—, and n is between 20 and 35.

The fixing component of the treatment solution is a fixative including,but not limited to, an aldehyde, oxazolidine, alcohol, cyclic urea, orthe like. Suitable examples include, without limitation, formaldehyde,glutaraldehyde; diazolidinyl urea, imidazolidinyl urea, dimethylol urea,dimethylol-5,5-dimethylhydantoin, 2-bromo-2-nitropropane-1,3-diol;quaternary adamantine; -hydroxymethyl -1-aza-3,7-dioxabicyclo(3.3.0)octane and 5-hydroxymethyl-1-aza-3,7-dioxabicyclo (3.3.0)octaneand 5-hydroxypoly-methyleneoxy-methyl-1-aza-3,7-dioxabicyclo(3.3.0)octane, sodium hydroxymethyl glycinate, and mixtures thereof. Ina preferred embodiment, an aldehyde fixative, such as paraformaldehyde,formaldehyde or glutaraldehyde is used.

Other fixatives may be used, such as those disclosed in U.S. Pat. Nos.5,196,182, 5,262,327, 5,460,797, 5,811,099, 5,849,517, 6,221,668,5,529,933, and 6,187,590, all of which are hereby incorporated byreference.

Example 1 illustrates an example of preparing the nucleated red bloodcell component (NRBC analog) using alligator red blood cells. As shown,using the method and the treatment solution of the present invention,the size, optical property, as well as fluorescence property of thetreated cell nuclei are altered from their natural values, orproperties. Herein the fluorescence property refers to the fluorescencesignals measured on a flow cytometer upon staining the nucleated bloodcells, or the treated cell nuclei with a nucleic dye, On the other hand,the optical property can be affected by size and granularity of thenuclei. More specifically, the NRBC analog obtained using the specifictreatment solution composition shown in Example 1 is smaller in size,more light scattering, and less fluorescent as shown in FIGS. 2A thru2E, in comparison to the untreated nucleated red blood cells undervarious measurement conditions.

Example 2 illustrates another example of preparing the NRBC analog usingalligator red blood cells, wherein the process steps of preparing theNRBC analogs are the same as in Example 1, however, the treatmentsolutions are different in terms of the conditioning component. InExamples 1, the conditioning component is a disodiumhydrogenphosphate/sodium dihydrogenphosphate buffer system(Na₂HPO₄/NaH₂PO₄), and the treatment solution has an osmolarity of 390mOsm. In Example 2, the conditioning component includes ammonium citrateand tetramisole, and the treatment solution has an osmolarity of 560mOsm. As shown in FIGS. 4A thru 4E, the obtained NRBC analog using thetreatment solution of Example 2 has substantially smaller sizes, and hasa stronger fluorescence signal than the analog obtained using thetreatment solution used in Example 1.

Although the exact reaction mechanism is not fully understood, it can beappreciated that when in contact with the nucleated red blood cells, thethree functional components of the treatment solution act simultaneouslyon the cells, and the cell nuclei. The lytic component enablespermeation of the treatment solution into the cell membrane and allowingthe treatment solution in contact with the cell nuclei. The lyticcomponent can further permeate the nucleus membrane. At the same time,the fixing component, in a competing mode, preserves the cell nuclei byfixation. On the other hand, the conditioning component provides areaction condition with certain pH and osmolarity, which affects thecell and cell nucleus responses to the lytic and fixing components. Incombination, such treatment condition has resulted in alterations ofnucleus properties, which can be utilized for simulating NRBCs of ablood sample on a blood analyzer.

In a further embodiment, the reference control composition furtherincludes a white blood cell component which simulates a white blood cell(WBC) under a specific detection condition. In the presence of a whiteblood cell component, a ratio between the NRBC component and the whiteblood cell component can be used to report the numbers of NRBCs per 100WBC, which is the same unit used for reporting nucleated red blood cellsin a blood sample in clinical laboratories. Preferably, the white bloodcell component or analog has properties similar to one major white bloodcell subpopulation, such as granulocytes or lymphocytes.

Furthermore, the white blood cell component can include more than onewhite blood cell subpopulation component or analog, for example, two,three, four or five white blood cell analogs to simulate the white bloodcell subpopulations for a differential analysis. Suitable examples ofwhite blood cell analogs include stabilized and fixed mammalian whiteblood cells, and processed and/or fixed human and animal red bloodcells, as known in the art. In one embodiment, the white blood cellanalogs can be made from processed goose and alligator red blood cellsfor differential analysis using a combination of impedance and lightscatter measurement, as taught in U.S. Pat. Nos. 5,320,964 and5,512,485, which are herein incorporated by reference in their entirety.In another embodiment, the white blood cell analogs can be made fromprocessed avian and human red blood cells for differential analysisusing an impedance measurement, as taught in U.S. Pat. No. 4,704,364,which is herein incorporated by reference in its entirety. In a furtherembodiment, the white blood cell analogs can be made from fixedmammalian white blood cells. The mammalian white blood cells are fixedprior to lysing the red blood cells in the whole blood during thepreparation of the white blood cell analogs.

Optionally, the mammalian white blood cells and the animal red bloodcells can be further treated by contacting with a lipoprotein during theprocess of preparing the white blood cell analogs. The contact withlipoprotein can occur prior to fixing the white or red blood cells, itcan also occur after fixing and during storage in the suspension medium,as taught in U.S. Pat. Nos. 5,320,964, 5,512,485, 6,406,915, 6,403,377,6,399,388, 6,221,668, and 6,200,500, which are incorporated herein byreference in their entirety.

In another embodiment, the present invention provides a referencecontrol composition which comprises the above described nucleated redblood cell component, a white blood cell component, and additionally ared blood cell component and a platelet component in the suspensionmedium.

The red blood cell component can be stabilized human or animal red bloodcells, preferably, stabilized human red blood cells. The process ofmaking the red blood cell component has been described in detail in U.S.Pat. Nos. 4,299,726 and 4,358,394, which are incorporated by referencesin their entirety. The platelet component can be stabilized human oranimal platelets, or platelet analogs made from other cell types. Onesuitable example is processed goat red blood cells as the plateletanalog, as disclosed in U.S. Pat. Nos. 4,264,470, 4,389,490 and4,405,719, which are incorporated by references in their entirety.

The red blood cells of a blood sample or the stabilized human red bloodcells in the reference control composition are lysed under lysingconditions normally used for preparing a blood sample for themeasurement of nucleated red blood cells and white blood cells, andshould not be detected in the measurement if the analyzer operatesproperly. The platelets of a blood sample under the lysing conditionsare reduced in size and they are either below the detection thresholdfor the measurement of nucleated red blood cells, or are separated fromthe nucleated red blood cells. The platelet analog described abovesimulates the response of the platelets of a blood sample under thelysing condition. Therefore, the red blood cell component and plateletcomponent in the reference control composition further reflect theresponse of the control composition to the lysing reagent, as well asthe reaction conditions on the instrument. Hence, the reference controlcomposition containing red blood cell and platelet components canprovide further information related to instrument operating conditions.

Moreover, the reference control composition containing red blood celland platelet components can also be used for the red blood cell andplatelet measurements, which are commonly performed together with themeasurements of the white blood cells and nucleated red blood cells onan automated hematology analyzer.

Optionally, the reference control composition can further comprise areticulocyte component for the analysis of reticulocytes.

Example 3 illustrates a preparation of a reference control compositioncontaining a nucleated red blood cell component made with the processdescribed in Example 1 a white blood cell component, a red blood cellcomponent and a platelet component.

Example 4 further illustrates a preparation of a reference controlcomposition containing a nucleated red blood cell component, a pluralityof white blood cell subpopulation components, a red blood cell componentand a platelet component.

The reference control composition containing the prepared nucleated redblood cell component can be utilized for nucleated red blood cellmeasurement using various measurement methods.

In one embodiment, the present invention provides a method of using thereference control containing the nucleated red blood cell component forthe measurement of nucleated red blood cell using a DC impedancemeasurement. The measurement method and instrumentation used formeasuring nucleated red blood cells in a blood sample were described inU.S. Pat. No. 6,410,330 which is herein incorporated by reference in itsentirety.

As shown in FIGS. 2A and 4A, the NRBC analogs made using TreatmentSolutions 1 and 2 with the process described in Example 1 simulated thesize of nucleated red blood cells of human whole blood samples whenprocessed with a lytic reagent and measured by DC impedance measurementon a hematology analyzer, as described in detail in Example 1.

In another embodiment, the present invention provides a method of usingthe reference control containing the nucleated red blood cell componentfor the measurement of nucleated red blood cell using opticalmeasurements. The optical measurements include light scattermeasurement, axial light loss measurement, and fluorescence measurement.

The analysis of NRBCs using optical measurement is performed in afocused-flow flow cell. When a particle such as a blood cell, passesthrough the aperture of the flow cell, it scatters the incident light inall directions. The light scatter signals can be detected by a lightdetector at various angles relative to the incident light beam between0° to 180°. The light scatter signals detected in less than 10° from theincident light are commonly called low angle light scatter. The lightscatter signals detected from about 10° to about 70° from the incidentlight are called medium angle light scatter, and the light scattersignal detected at about 90° of the incident light are calledright-angle light scatter, or side scatter. On a flow cytometer thelight scatter signals detected at less than 20° from the incident lightare commonly referred to as forward light scatter. The characteristicsof light scatter signals are affected by the size of a cell, thecontents of a cell, and the surface properties of a cell.

Axial light loss (ALL, also known as forward extinction) is generallythe decrease in light energy due to a particle passing through a beam ofincident light and being detected by a photodetector. When the beam ofincident light strikes a particle, the light is either scattered orabsorbed, both of which remove energy from the incident light and theincident beam is attenuated. This attenuation is referred to asextinction. When viewed along the axis of the beam of incident light, itis referred to as axial light loss. Generally ALL signals are detectedat an angle from about 0° to about 1° from the incident light. ALLsignals are strongly influenced by the size of the cell.

Furthermore, nucleated blood cells upon staining by a nuclear dye can bemeasured by fluorescence measurement. Depending on the dyes used, thefluorescence signals at several different wavelengths can be utilizedfor differentiation of nucleated blood cells. It has been found thatnucleated red blood cells have different light scattering, axial lightloss and fluorescence properties, either significant or minor, fromother cell types, which have been utilized for differentiation of thisabnormal blood cell population from other cell types.

One suitable example of the measurement method and instrumentation withwhich the NRBC analog made by the method of the present invention can beutilized as a reference control has been described in detail in U.S.Pat. Nos. 5,874,310 and 5,917,584, which are herein incorporated byreference in their entirety. More specifically, two angles of lightscatter signals are used for differentiation of nucleated red bloodcells from other cell types. One of the light scatter is a low anglelight scatter signal which is less than 10°. The second light scatterangle is a low, a medium or a right-angle light scatter signal.Furthermore, a light scatter measurement in combination of a DCimpedance measurement can also be used for measuring nucleated red bloodcells.

A further suitable example of the measurement method and instrumentationwith which the NRBC analog made by the method of the present inventioncan be utilized as a reference control has been described in detail inthe co-pending patent application Ser. No. 11/048,086, which is hereinincorporated by reference in its entirety. In this methods axial lightloss and low angle light scatter measurements, preferably from about 3°to 7° or axial light loss and DC impedance measurements, or combinationthereof are used for differentiation of nucleated red blood cells fromother cell types.

Moreover, as shown in FIGS. 2E and 4E, the NRBC analogs made usingTreatment Solutions 1 and 2 with the process described in Example 1 canbe used to simulate the fluorescence properties of the nucleated redblood cells of human whole blood sample when processed with a reagentsystem and measured by fluorescence measurements at 525 nm and 675 nm ona flow cytometer. The process of analysis of NRBC in a blood sample on aCoulter FC 500 flow cytometer is described in Example 1 and illustratedin FIGS. 3C and 3D.

In yet another embodiment, the present invention provides a method ofusing the reference control containing a nucleated red blood cellcomponent for the measurement of nucleated red blood cell using acombination of DC impedance and a VCS measurement method. Themeasurement method and instrumentation used for the measurement aredescribed in U.S. Pat. No. 6,472,215, which is herein incorporated byreference in its entirety. The term of VCS measurement is referred to amultiple parameter measurement of DC impedance, radio frequencyimpedance and medium angle light scatter signals, as described in U.S.Pat. No, 5,125,737, which is herein incorporated by reference in itsentirety.

As shown in FIGS. 2B, 2C, 4B and 4C, the NRBC analogs made usingTreatment Solutions 1 and 2 with the process described in Example 1 canbe used to simulate the nucleated red blood cells of human whole bloodsample when processed with a reagent system and measured by acombination of DC impedance and VCS measurements on a hematologyinstrument. The process of analysis of NRBC on a Coulter LH750hematology analyzer is described in detail in Example 1.

The following examples are illustrative of the invention and are in noway to be interpreted as limiting the scope of the invention, as definedin the claims. It will be understood that various other ingredients andproportions may be employed, in accordance with the proceedingdisclosure.

EXAMPLE 1 Preparation of NRBC Analog Using Alligator Red Blood Cells

The following reagent compositions were prepared for the preparation ofthe NRBC analog.

Phosphate Buffered Saline Solution (PBS) Sodium dihydrogenphosphate: 0.2g Disodium hydrogenphosphate 7H₂O: 2.0 g Sodium azide: 0.1 g Sodiumchloride: 9.4 g Qs to 1 liter with distilled water: pH approximately 7.4osmolality 315 to 345 mOsm/kg H₂O

Treatment Solution 1 Disodium hydrogenphosphate 7H₂O: 4.781 g Sodiumdihydrogenphosphate: 0.209 g 6% Paraformaldehyde: 0.6 ml 1% PMSF in DMSO(10 mg/ml) solution: 0.1 ml Tetradecyltrimethylammonium bromide 0.125 gIgepal SS-837 (Rhone-Poulenc) 0.075 ml Plurofac A38 prill surfactant(BASF Corp.) 0.02 g Qs to 1 liter with distilled water: pH approximately7.6 osmolarity 390 mOsm/kg H₂O Note: PMSF is α-Toluenesulfonyl fluoride.Igepal SS-837 is an ethoxylated phenol. Plurofac A38 is an ethoxylatedalkyl alcohol.

Suspension Medium 1 Range Preferred Component (g/liter) (g/liter)Xanthine compound   1-10 2-7 Adenosine monophosphate 0.1-1.0 0.2-0.8Inosine 0.1-1.0 0.2-0.8 pH adjusting agents pH 5.8-6.8 pH 6.0-6.5sufficient to obtain Osmolality adjusters 200-400 mOsm 250-350sufficient to obtain Preservative effective amount 2.0-6.0 Qs to 1 literwith distilled water

Suspension Medium 2 Preferred (g or ml/liter) Propyl paraben 0.3 to 1.0g Methyl paraben 0.5 to 1.0 g Procaine hydrochloride 0.1 to 0.5 gDeoxycholic acid 0.1 to 0.9 g Lactose 10.0 to 50.0 g Actidione 0.1 to0.6 g Trisodium citrate dehydrate 3.0 to 8.0 g Citric acid monohydrate0.3 to 0.9 g Sodium dihydrogenphosphate 0.8 to 2.5 mg monohydratePhenergan hydrochloride 0.1 to 1.0 g Colistimethate, sodium 0.2 to 0.9 gPenicillin G., sodium 0.5 × 10⁶ to 3 × 10⁶ units Kanamycin sulfate 0.2to 0.8 g Neomycin sulfate 0.2 to 1.0 g 5′-AMP 0.4 to 1.0 g Adenine 0.2to 0.8 g Inosine 0.4 to 1.0 g Dihydrostreptomycin sulfate 0.2 to 1.0 gTetracycline hydrochloride 0.2 to 1.0 g 30% Bovine albumin 100 to 350 mlQs to 1 liter with distilled water

Suspension Medium 3 Preferred (g or ml/liter) Sodium Chloride 0.18 gSodium Hydroxide Pellets 0.05 g B.S.A. Protease-free 12.0 g Pluronic F680.2 g Sodium Phosphate monobasic 0.18 g EDTA, Di-sodium 0.02 g Lactose2.5 g Citric acid (anhydrous) 0.032 g Neomycin Sulfate 0.05 gAdenosine's-monophosphate 0.05 g TRI-sodium citrate 0.5 g Sodiumpenicilin G 0.08 g Inosine 0.05 g Procaine HCL 0.032 g Phenergan HCL0.01 g 5-Fluorouracil 0.1 g MOD-U-CYTE VIII 20.0 ml Imidazole 0.025 g Qsto 1 liter with distilled water pH 6.9-7.1 osmolarity 330-410 mOsm/kgH₂O

Process steps for preparing NRBC analog:

-   -   1. Collect a quantity of alligator whole blood in an        anticoagulant containing container. Centrifuge the alligator        whole blood and remove the top layer (including white blood        cells, platelets and plasma).    -   2. Wash the packed alligator red blood cells three times with        the phosphate buffered saline solution.    -   3. Wash the packed alligator red blood cells with the Suspension        Medium 1 and re-suspend washed packed cells in the Suspension        Medium 1. Preferably, the cell count is in a range from about        0.4×10⁶ to about 0.6×10⁶ cell/μl.    -   4. Treat the re-suspended alligator red blood cells by adding a        predetermined volume of the re-suspended cells to an equal        volume of the Treatment Solution 1, and mix well by inversion to        form a cell treatment suspension.    -   5. Incubate the cell treatment suspension at 4° C. overnight    -   6. Remove the cell treatment suspension from refrigerator and        centrifuge at 1000 rpm for 15 minutes, and remove the        supernatant.    -   7. Re-suspend the treated cells in the Suspension Medium 3 for        analysis on a blood analyzer.

The NRBC analog obtained from the above-described treatment process wasanalyzed on a Coulter® LH750 hematology analyzer (manufactured byBeckman Coulter, Inc., California). The Coulter LH750 hematologyanalyzer has a RBC bath using three non-focused-flow apertures and afirst DC impedance detector for measuring red blood cells; a WBC bathusing three non-focused-flow apertures and a second DC impedancedetector for measuring white blood cell count, and a 3-part differentialanalysis of white blood cells and nucleated red blood cells; and afocused-flow flow cell with a VCS detection system for a 5-partdifferential analysis of white blood cells and nucleated red bloodcells. The VCS detection system measures DC impedance, radio frequencyimpedance and medium angle light scatter signals of a cell passingthrough the flow cell. Herein the 3-part differential analysis and5-part differential analysis of white blood cells refer to thedifferential analysis of white blood cells into three and fivesubpopulations, respectively.

A blood sample or a reference control was aspirated by the Coulter LH750hematology analyzer. A first aliquot of the sample was diluted with anisotonic blood diluent, LH700 Series Diluent (product of BeckmanCoulter, Inc.), to form a first sample mixture. The first sample mixturewas drawn through a set of three apertures by a vacuum source. Eachblood cell was measured, as it passed through the apertures by the firstDC impedance detector to obtain red blood cell parameters. A secondaliquot of the blood sample was diluted with the LH700 Series Diluent,then mixed with a volume of a first lytic reagent, Lyse S® III diff(product of Beckman Coulter, Inc.), to form a second sample mixture. Thesecond sample mixture was drawn through a set of three apertures by avacuum source, and measured by the second DC detector to obtain whiteblood cell count, 3-part differential analysis of white blood cells andanalysis of nucleated red blood cells. A third aliquot of the sample wasmixed with a volume of a second lytic reagent, Erythrolyse™ II, to lysered blood cells and subsequently mixed with a volume of a stabilizingreagent, StabiLyse™, (both are products of Beckman Coulter, Inc.), toform a third sample mixture. The third sample mixture was delivered tothe focused-flow flow cell for a 5-part differential analysis of whiteblood cells and analysis of nucleated red blood cells. The measurementwas performed at a temperature in the range of about 18° to about 28° C.The data obtained from measurements of the second and the third samplemixtures were analyzed collectively to provide the report of thenucleated red blood cells.

FIG. 1A shows a red blood cell distribution histogram (the first samplemixture in the RBC bath) obtained from washed alligator red blood cells(only processed from Steps 1 to 3, without treatment by TreatmentSolution 1). As shown, the washed alligator red blood cells were verylarge, having a cell size above 300 fl. FIG. 1B shows a partiallydisplayed white blood cell distribution histogram (the second samplemixture in the WBC bath) of the washed alligator red blood cells. Asshown, under the lysing condition the nucleated red blood cells measuredin the WBC bath were substantially at their nuclear size, having a verybroad distribution with a peak at about 55 fl. It is noted that therewas a substantial amount of cell debris on the left-most region of thehistogram, resulting from the lysing reaction. FIG. 1C shows a DC vs.RLS (rotated light scatter) scattergram of the washed alligator redblood cells. The nucleated red blood cells measured under the lysingcondition of the third sample mixture located at the bottom of the DCaxis and around the center of the RLS axis. Herein, the RLS is afunction of DC and the medium angle light scatter of the VCS detectionsystem.

FIG. 2A shows a red blood cell distribution histogram of a NRBC analogmade of treated alligator red blood cells with the process describedabove using Treatment Solution 1. As shown, the NRBC analog had a sizeclose to the nucleus size of the alligator red blood cells under thelysing condition, which is shown in FIG. 1B. FIG. 2B shows the partiallydisplayed white blood cell distribution histogram of the NRBC analogunder the lysing condition in the WBC bath. As shown, the NRBC analogwas smaller in size (about 45 fl) and has a narrower and well-definedcell distribution, in comparison to the washed alligator red bloodcells. FIG. 2C shows a DC vs. RLS scattergram of the NRBC analog underthe lysing condition used for the third aliquot of the blood sample.

FIG. 3A shows a white blood cell distribution histogram of a clinicalblood sample containing 27 NRBC/100 WBC, analyzed on a Coulter LH750hematology analyzer using the same process described above. FIG. 3Bshows the DC vs. RLS scattergram of the clinical sample. It is notedthat the display scale of the histogram in FIG. 3A is different fromthat in FIGS. 1B and 2B, wherein FIG. 3A shows the entire white bloodcell distribution, FIGS. 1B and 2B only show the left-most region wherethe NRBC population located. It is apparent that the NRBC analog shownin FIGS. 2B and 2C was similar in size to human nucleated red bloodcells under the same sample analysis conditions.

The NRBC analog obtained from the above-described treatment process wasalso analyzed on a Coulter FC 500 flow cytometer (manufactured byBeckman Coulter, Inc., California). A 25 μl of a blood sample or areference control was mixed with 10 μl of FITC labeled anti-CD45antibody and incubated at room temperature for 30 minutes for labelingthe white blood cells. The incubated sample was then mixed with 250 μlof a hypotonic acid lysing reagent (Solution A, which contained anucleic acid dye) and allowed to stand for 30 seconds to lyse the redblood cells, then mixed with 500 μl of hypertonic alkaline reagent(Solution B) and allowed to stand for 5 minutes at room temperature torestore osmolarity and neutralize the pH and to form a sample mixture.The sample mixture was aspirated by the Coulter FC 500 flow cytometerand measured by the forward and side light scatter detectors, andfluorescence detectors at 525 nm (FL1) and 675 nm (FL4). Solution Acomprises 2.10 g/l citric acid monohydrate, 0.56 g/l disodiumhydrogenphosphate and 100 mg/l of propidium iodide in de-ionized water,and has a pH of 3.0 and osmolarity of 16 mOsm/kg H₂O. Solution Bcomprises 0.95 g/l sodium dihydrogenphosphate dehydrate, 6.24 g/ldisodium hydrogenphosphate and 10.2 g/l of sodium chloride in de-ionizedwater, and has a pH of 7.5 and osmolarity of 420 mOsm/kg H₂O. Thisanalysis method has been described in detail by Tsuji et al, Cytometry37:291-301, 1999: which is herein incorporated by reference in itsentirety.

FIG 1D shows a FS (forward light scatter) vs. SS (side scatter)scattergram, and FIG. 1E shows a FL4 (log) vs. FL1 (log) scattergram, ofthe washed alligator red blood cells (washed as described previously).As shown, the washed alligator red blood cells appear at the bottom leftcorner of the FS vs. SS scattergram, substantially at their nuclearsize. In the FL4 (log) vs. FL1 (log) scattergram, the nucleated redblood cells appear in the left upper quadrant. These cells had strongFL4 signals, which are also referred to as PI^(bright). They were CD45⁻,indicated by their weak FL1 signals.

FIGS. 2D and 2E show the FS vs. SS scattergram and the FL4 vs. FL1scattergram, respectively, of the NRBC analog made of the treatedalligator red blood cells as described above using Treatment Solution 1.As shown, the NRBC analog had increased light scatter signals,particularly the side scatter signal. On the other hand, the NRBC analoghad a decreased FL4 signal. It is noted that although the NRBC analoghad relatively weaker FL4 signals than the washed alligator red bloodcells, the signals were sufficient for simulating the blood samples.Furthermore, the different distribution characteristics of the NRBCanalog can be used as a fingerprint of the reference control, which hascertain advantages for the instrument to recognize a reference controlfrom a blood sample.

FIGS. 3C and 3D show the FL4 vs. FL1 scattergram of a normal whole bloodsample and a clinical sample containing 13.7 NRBC/100 WBC, respectively.As shown in FIG. 3D, the NRBC population located in the left upperquadrant of the scattergram. As shown in FIG. 3C, the normal bloodsample did not have a cell population located in this region. Therefore,the NRBC analog made using the process described above can be used forsimulating human NRBCs using fluorescent measurements.

The NRBC analog was also made with the above-described process stepsexcept using Suspension Medium 2 in step 3. The analyses on the CoulterLH750 hematology analyzer and the Coulter FC 500 flow cytometer showedthat the NRBC analog made with Suspension Medium 2 was similar to thosemade with Suspension Medium 1.

EXAMPLE 2 Preparation of Nucleated Red Blood Cell Component UsingAlligator Red Blood Cells

An amount of the same alligator whole blood used in Example 1 wastreated with the same process steps described in Example 1, except usingTreatment Solution 2 shown below.

Treatment Solution 2 Ammonium citrate tribasic: 19.8 g Tetramisole: 1.8g 6% Paraformaldehyde: 0.6 ml 1% PMSF in DMSO (10 mg/ml) solution: 0.1ml Tetradecyltrimethylammonium bromide 0.125 g Igepal SS-837(Rhone-Poulenc) 0.075 ml Plurofac A38 prill surfactant (BASF Corp.) 0.02g Qs to 1 liter with distilled water: pH approximately 5.66 osmolarity560 mOsm/kg H₂O

It is noted that the lytic component and the fixing component were thesame between Treatment Solution 1 and Treatment Solution 2, however, theconditioning component was different, which resulted in different pH andosmolarity between the two treatment solutions.

The NRBC analog made using Treatment Solution 2 was analyzed on theCoulter LH750 hematology analyzer and the Coulter FC 500 flow cytometer,using the same sample analysis procedure described above.

FIG. 4A shows the red blood cell distribution histogram of the NRBCanalog made using Treatment Solution 2. As shown, this NRBC analog had asubstantially smaller size than that of the NRBC analog obtained inExample 1. FIG. 4B shows the partially displayed white blood celldistribution histogram of this NRBC analog under the lysing condition inthe WBC bath, which was also smaller than that of the NRBC analogobtained in Example 1. However, both NRBC analogs were similar in sizeto human nucleated red blood cells. FIG. 4C further shows the DC vs. RLSscattergram of the NRBC analog.

FIGS. 4D and 4E show the FS vs. SS and FL4 vs. FL1 scattergrams,respectively, of the NRBC analog of Example 2. As shown in FIG. 4D, theNRBC analog of Example 2 had an increased forward light scatter signal,however, decreased side scatter signals. As shown in FIG. 4E, the NRBCanalog had fluorescence signals very similar to that of human nucleatedred blood cells.

EXAMPLE 3 Reference Control Composition Containing a Nucleated Red BloodCell Component, a White Blood Cell Component, and Red Blood Cell andPlatelet Components

Procedure:

-   -   1. Provide a predetermined volume of the Suspension Medium 1        described in Example 1.    -   2. Add a predetermined amount of stabilized human red blood        cells in the suspension medium. The stabilized human red blood        cells were prepared following the procedure described in U.S.        Pat. Nos. 4,299,726 and 4,358,394.    -   3. Add a predetermined amount of platelet analog in the        suspension medium containing the stabilized human red blood        cells. The platelet analog is made of fixed goat red blood cells        following the procedure described in U.S. Pat. Nos. 4,264,470,        4,389,490 and 4,405,719.    -   4. Add a predetermined amount of fixed goose red blood cells as        the white blood cell component into the suspension medium        containing the stabilized human red blood cells and platelet        analog.    -   5. Add a predetermined amount of NRBC analog prepared in Example        1 or 2 into the suspension medium containing the stabilized        human red blood cells, platelet analog and white blood cell        component.    -   6. Mixing the reference control composition formed in step 5.        The cell concentration of the red blood cell, white blood cell        and platelet components are prepared to simulate the        corresponding cell concentrations of a human whole blood sample.        The cell concentration of the NRBC analog is prepared to        simulate a clinical sample containing a certain level of        nucleated red blood cells, preferably in a range of 1 to 50 NRBC        per 100 WBC.

EXAMPLE 4 Reference Control Composition Containing a Nucleated Red BloodCell Component, Multiple White Blood Cell Subpopulation Components andRed Blood Cell and Platelet Components

The procedure for making this reference control composition isessentially the same as the procedure described above in Example 3,except that in step 4 predetermined amounts of multiple white blood cellsubpopulation analogs are added into the suspension medium containingthe stabilized human red blood cells and platelet analog.

The multiple white blood cell subpopulation analogs are preparedfollowing the procedures described in U.S. Pat. Nos 4,704,364, 5,320,964and 5,512,485. Using the multiple white blood cell subpopulation analogsprepared following the procedures described in U.S. Pat. No. 4,704,364,the control composition can be used for nucleated red blood cellmeasurement and differentiation of white blood cells into threesubpopulations. Using the multiple white blood cell subpopulationanalogs prepared following the procedures described in U.S. Pat. Nos.5,320,964 and 5,512,485, the reference control composition can be usedfor nucleated red blood cell measurement and differentiation of whiteblood cells into five subpopulations.

While the present invention has been described in detail and pictoriallyshown in the accompanying drawings, these should not be construed aslimitations on the scope of the present invention, but rather as anexemplification of preferred embodiments thereof. It will be apparent,however, that various modifications and changes can be made within thespirit and the scope of this invention as described in the abovespecification and defined in the appended claims and their legalequivalents. All patents and other publications cited herein areexpressly incorporated by reference.

1. A reference control containing a nucleated red blood cell componentcomprising: (a) a nucleated red blood cell component obtained bytreating a blood cell that contains a nucleus with a treatment solutioncomprising a conditioning component, a lytic component and a fixingcomponent in one step, and allowing three said components tosimultaneously interact with said nucleus, thereby causing alteration ofsize and optical property of said nucleus from a natural value to atarget value suitable for simulating nucleated red blood cells of ablood sample on a blood analyzer; and (b) a suspension medium suitablefor delivering said nucleated red blood cell component to said bloodanalyzer for analysis of said nucleated red blood cells.
 2. Thereference control of claim 1, wherein said optical property is lightscatter property, axial light loss property, or fluorescence property ofsaid nucleus upon staining with a fluorescence dye.
 3. The referencecontrol of claim 1 further comprising a white blood cell component. 4.The reference control of claim 3, wherein said white blood cellcomponent comprises a plurality of subpopulation components forsimulating a plurality of white blood cell subpopulations.
 5. Thereference control of claim 3 further comprising one or more componentsselected from the group consisting of red blood cell component, plateletcomponent, and reticulocyte component.
 6. The reference control of claim1, wherein said blood cell comprises reptilian, avian or fish red bloodcells.
 7. A method of making a reference control containing a nucleatedred blood cell component comprising the steps of: a) providing a bloodcell containing a nucleus; b) treating said blood cell with a treatmentsolution comprising a conditioning component, a lytic component and afixing component in one step, and allowing three said components tosimultaneously interact with said nucleus, thereby causing alteration ofsize and optical property of said nucleus from a natural value to atarget value suitable for simulating nucleated red blood cells on ablood analyzer; and c) suspending treated blood cell obtained from step(b) in a suspension medium to form said reference control.
 8. The methodof claim 7, wherein said treating said blood cell is mixing said bloodcell with said treatment solution to form a cell treatment suspensionand incubating said cell treatment suspension for a period of from about15 minutes to about 24 hours.
 9. The method of claim 8, wherein saidincubating said cell treatment suspension is at about 4° C.
 10. Themethod of claim 7, wherein said treatment solution is hypertonic.
 11. Amethod of using a reference control containing a nucleated red bloodcell component comprising the steps of: a) providing a reference controlcontaining a nucleated red blood cell component obtained by treating ablood cell that contains a nucleus with a treatment solution comprisinga conditioning component, a lytic component and a fixing component inone step, and allowing three said components to simultaneously interactwith said nucleus, thereby causing alteration of size and opticalproperty of said nucleus from a natural value to a target value suitablefor simulating nucleated red blood cells of a blood sample; b) providinga blood analyzer adapted for analyzing said blood cell sample anddifferentiating nucleated red blood cells from other cell types; c)passing said reference control through said blood analyzer for detectionof said nucleated red blood cell component; and d) reporting nucleatedred blood cells in said reference control.
 12. The method of claim 11,wherein said differentiating nucleated red blood cells is obtained usingan impedance measurement.
 13. The method of claim 11, wherein saiddifferentiating nucleated red blood cells is obtained using an opticalmeasurement.
 14. The method of claim 13, wherein said opticalmeasurement is one or more measurements from the group consisting offluorescence, light scatter and axial light loss measurements.
 15. Themethod of claim 11, wherein said differentiating nucleated red bloodcells is obtained using a combination of impedance and opticalmeasurements.